An estimated 285 million people, corresponding to 6.4% of the world's adult population is affected by diabetes mellitus. The number is expected to increase to 438 million worldwide by 2030, corresponding to 7.8% of the adult population. More than 50 million individuals are affected in Europe. The majority (around 85%) of patients have type 2 diabetes mellitus (T2D), while some 10-15% of the patients suffer from type 1 diabetes mellitus (T1D). It is one of the major causes of premature illness and death worldwide. Diabetes causes severe long-term complications and psychosocial problems, imposing a heavy burden of morbidity and premature mortality. Diabetes is the leading cause of blindness and visual disability, limb amputation, end-stage renal failure and neuropathy. It is associated with a greatly increased incidence of cardiovascular disease, including stroke, myocardial infarction and heart failure. Cardiovascular disease accounts for more than 50% of all deaths among diabetic patients in Europe. Because T2D is increasingly prevalent and developing earlier in life, increasing the duration of the disease by decades, many more people are developing severe diabetic complications, and suffering decreases their life quality and expectancy.
Diabetes incurs many costs, and is particularly expensive because it is life-long and causes major medical problems. These costs include direct costs from physician and nursing services, hospital services, laboratory services, drugs, education and training of the patients and indirect costs from time lost from work, chronic nursing and general socio-economic support, early retirement, protracted morbidity and premature mortality.
Overall costs of diabetes mellitus, comprising both direct and indirect costs, have been calculated in various countries and are enormous. For instance, the cost of treating one patient over a 25-year period is in the range of 100,000-200,000 EURO.
The pancreas, an organ about the size of a hand, is located behind the lower part of the stomach. It comprises two structures that are both morphologically and physiologically different: the exocrine pancreas, which produces the enzymes involved in digestion (amylase, lipase, etc.) and sodium bicarbonate, and the endocrine pancreas, which produces the hormones involved in the control of blood glucose (insulin, glucagon, somatostatin and pancreatic polypeptide). The cells of the endocrine pancreas are organized as micro-organs dispersed in the pancreas in the form of islets (islets of Langerhans or pancreatic islets). Each pancreatic islet is made up of 4 cell types: alpha cells, beta cells, delta cells and PP cells. In rats, the alpha cells are located at the periphery of the islet and secrete glucagon. The beta cells are found at the center of the islet and are the only cells capable of secreting insulin in response to glucose. The delta cells are at the periphery and secrete somatostatin. In humans, the cells are scattered inside the Langerhans islets. The function of the PP cells is more controversial (synthesis of pancreatic polypeptide).
Insulin is a hormone that helps the body to use glucose for energy. Diabetes develops when the body does not make enough insulin, cannot use insulin properly, or both, causing glucose to build up in the blood. In type 1 diabetes mellitus (T1D)—an autoimmune disease—the beta cells of the pancreas no longer make insulin because the body's immune system has attacked and destroyed them. A person who has T1D must take insulin daily to live. Type 2 diabetes mellitus (T2D) usually begins with a condition called insulin resistance, in which the body has difficulty using insulin effectively. Over time, insulin production declines as well, so many people with T2D eventually need to take insulin. In addition, a condition called hyperinsulinemia occurs in patients with increased beta cell populations when compared to healthy subjects. Patients with hyperinsulinemia are at high risk of developing seizures, mental retardation, and permanent brain damage. Since glucose is the primary substrate used by the CNS, unrecognized or poorly controlled hypoglycemia may lead to persistent severe neurologic damage. Transient hyperinsulinemia is relatively common in neonates. An infant of a diabetic mother, an infant who is small or large for gestational age, or any infant who has experienced severe stress may have high insulin concentrations.
Among the treatments of diabetes, besides the regular administration of insulin, one of the approaches for the physiological control of glycemia and for normalization of glycemia in diabetics is to restore insulin secretion in vivo from cells. Several strategies have been proposed: xenotransplantation of insulin-producing cells from animals, in vitro differentiation of isolated stem cells into insulin-secreting cells and re-implantation thereof in the patient or allotransplantation of isolated pancreatic islets from another subject.
The lack of a cellular model for studying the beta cells, and also the lack of reliable and effective means of cell sorting suitable for this type of cells hinder the study of beta cell functioning and therefore the development of novel methods of treatment of type I and II diabetes.
The current attempts of imaging pancreatic beta cell mass are being done either by using MRI (magnetic resonance imaging) or by using PET (positron emission tomography) and SPECT (single photon emission computed tomography). In vivo imaging of beta cell mass needs a combination of very high sensitivity and high spatial resolution. MRI has the best spatial resolution but its major challenge is the low sensitivity achieved with magnetic probes and the ex-vivo labeling procedure currently used in islet transplantation. MRI has been successfully used by labeling of human islets with SPIO (small particles of iron oxide) ex vivo and subsequent islet transplantation (Evgenov et al 2006). Using this approach, it was possible to follow up the grafted beta cell mass up to 6 months after transplantation. This technique is, however, only usable for transplantation since it relies on ex vivo uptake of the marker by islet cells and cannot be used for in vivo imaging of beta cells in the pancreas. MRI was also used for tracking recruitment of diabetogenic CD8+ T-cells into the pancreas (Moore A et al, 2004), for detecting apoptosis in T1D progression using a Cy5.5 labeled annexin 5 probe (Medarova Z. et al, 2006) or for detection of micro vascular changes in T1D progression (Medarova Z. et al 2007), but the changes detected are semi-quantitative.
PET and SPECT have very high sensitivity and do not require ex-vivo labeling. On the other hand, these techniques have a lower spatial resolution as compared to MRI. PET or SPECT imaging is achieved using islet-specific receptor binding compounds or using compounds taken up specifically by transporters in the pancreatic islets labeled with radioactive tracers. Almost all current substrates used for beta cell PET/SPECT imaging bind or are taken up by the non-beta cells and, in some cases, even by exocrine cells in the pancreas. This results in the dilution of tracer and high backgrounds, making it currently impossible to quantify the beta cells which are scattered over the pancreas in tiny islets (100-300 [mu]m diameter) constituting only 1-2% of the total pancreas mass.
One of few beta-cell specific membrane proteins identified up to now is the zinc transporter ZnT8 protein encoded by the SLC30A8 gene (Chimienti F et al 2004, Seve et al 2004). ZnT8 co-localizes with insulin in the pancreatic beta cells (Chimienti et al 2006). Avalon (EP1513951 and WO03097802) and CEA (patent US2006246442, EP1563071 and WO2004046355) introduced patents on the fact that this protein was beta cell specific and on the usage of an antibody against ZnT8 for cancer therapy and for use in antibody test. Recently ZnT8 was identified as an autoantigen and the target of autoantibodies in type 1 diabetes (Wenzlau J M et al., 2007) and it is therefore not useful for beta cell detection.
The company Biogen-IDEC identified Kirrel 2 (filtrin or NEPH3), an immunoglobulin superfamily gene which is specifically expressed in the beta cells of pancreatic islet cells (Sun C. et al, 2003) and in kidney (Rinta-Valkama J et al 2007). Due to its very low expression levels this candidate is not useful for beta cell detection. Recently, it was shown that densin and filtrin can act as auto-antigens, and auto-antibodies against them are detected in T1D patients (Rinta-Valkama J et al 2007). However, this candidate was too lowly expressed to be of use in beta cell detection.
Tmem27 or collectrin was identified as a beta cell protein that stimulates beta cell proliferation (Fukui K et al, 2005) and which is cleaved and shed from the plasma membrane. However, collectrin expression is higher in the islet non beta cells than in the beta cells.
The free fatty acid receptor GPR40 (also called FFAR1) is a G-coupled receptor recently identified as islet specific and as a possible target for treatment of T2D (Bartoov-Shifman R et al 2007). This receptor, however, is expressed both in islet beta cells and in alpha cells (Flodgren E et al 2007), hampering its potential as a good beta cell biomarker.
PET imaging teams working on pancreas are also attempting to image beta cells. For this purpose, they are using compounds assumed to selectively bind or being taken up by islet-specific transporters and receptors. Examples of these compounds include glibenclamide, tolbutamide, serotonin, L-DOPA, dopamine, nicotinamide, fluorodeoxyglucose, and fluorodithizone. Glibenclamide and fluorodithizone are not specific enough to attain the robust signal to background ratio needed for quantification of beta cell mass via PET imaging. F-deoxy glucose (FDG) could not be used to successfully quantify beta cell mass (Malaisse W J et al. 2000, Ruf J et al. 2006, Nakajo M. et al., 2007) but could be used to discriminate between focal and diffuse hyperinsulinism (de Lonlay P et al 2005 and 2006, Otonkoski T et al 2006, Kauhanen S et al 2007, Ribeiro M J et al, 2007, Hardy OT., et al 2007).
The most promising compounds used up to now to image pancreatic beta cells are F18-DOPA and Dihydrotetrabenazine (DTBZ), both substrates being taken up by the VMAT2 transporter (Souza F. et al 2006, Simpson N R. et al. 2006), and Glucagon-like peptide 1 (GLP-1) or exendin, both ligands binding to the GLP-1 receptor (Gotthardt M. et al, 2002, Wild M. et al. 2006). Unfortunately, all the compounds mentioned above result in too high background levels and non-specific binding to various other intra-abdominal tissues such as kidney and liver.
As described above, the team of Paul Harris identified vesicular monoamine transporter 2 (VMAT2) and its ligand DTBZ as potential tools for beta cell imaging. DTBZ was labeled with C-11 and F-18 and a high pancreatic uptake was obtained in rodents and primates (Souza et al, 2006). Unfortunately, complete eradication of beta cells reduced the pancreatic uptake of DTBZ by only 30-40% showing that the compound lacks sufficient specificity for the beta cells (Kung et al 2007) to enable its use to assess beta cell mass.
Several auto-antibodies directed against insulin (K14D10), sulfatide (IC2), glutamic acid decarboxylase (GAD) or protein tyrosine phosphatase (IA2) have been identified. The team of Ian Sweet used a beta cell specific antibody (K14D10) and its Fab fragment for imaging/targeting beta cells but the antibody fragment with the best blood clearance failed to preferentially accumulate in the pancreas. The monoclonal antibody IC2 (Brogren C H et al 1986, Buschard K et al 1988), modified with a radioisotope chelator for nuclear imaging, showed highly specific binding and accumulation to beta-cells with virtually no binding to exocrine pancreas or stromal tissues (Moore A et al, 2001). Sulfatide, however, is also expressed in islet cell innervating Schwann cells and other neural tissues, which may hamper its use in beta cell imaging.
WO 2009/101181 discloses biomarkers located in the plasma membrane of pancreatic beta cells. These biomarkers are FXYD2 gamma isoforms a, b and c. FXYD2 is a regulating subunit of the Na, K-ATPase. The biomarkers are characterized by their 1) preferential expression in pancreatic islets as compared to surrounding tissues (total pancreas/exocrine tissue, liver, intestine, spleen, stomach) 2) higher expression in pancreatic beta cells than in pancreatic alpha cells or than in other islet non-beta cells 3) higher or comparable expression levels to glucokinase which is an enzyme specifically expressed in the pancreatic beta cell 4) location in the membrane and as such targetable with antibodies, peptides or small molecules, which allows imaging, targeting and immunohistochemistry and 5) expression is not induced during the process of inflammation of the beta cell mass and the protein is not enriched in T-cells and dendritic cells or in other cells participating in the inflammation process. WO 2009/101181 describes the use of antibodies for the detection of these biomarkers in order to allow the early identification of loss in beta cell mass and the follow up of therapies for diabetes, including islet transplantation, attempts at beta cell regeneration etc. Our experiments showed that the antibody described in WO 2009/101181 is not highly specific to the beta cells FXYD2 biomarker. The use of antibodies has several drawbacks. Indeed, besides their long manufacturing process, their specificity and affinity to the target might be low, especially for polyclonal antibodies. The accessibility of the target to heavy antibodies is reduced. Antibodies synthesized in animals (rabbits, rats) might cause an immune response when tested on humans. Moreover, antibodies are subject to degradation.
The present invention aims at providing a peptide that specifically binds a pancreatic beta cell marker, more precisely the FXYD2-gamma-a subunit, and avoiding all the mentioned disadvantages related to the use of antibodies. The invention provides also the use of the peptide in several applications which will be detailed hereafter.